Spotlight pubs.acs.org/acschemicalbiology
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TARGETING THE SOURCE OF ANTIBIOTIC RESISTANCE
As cancer cells spread, some wind up in poorly vascularized microenvironments where they adapt to conditions of high metabolic stress and become quiescent. These cells may be the culprits responsible for cancer recurrence after treatment has been completed, as they are often resistant to standard chemotherapies. Typically in cancer drug discovery screens, cancer cells are grown in culture as a monolayer, and various small molecules are tested for their ability to kill them. However, this model has inherent limitations, as it does not accurately recreate the three-dimensional and sometimes oxygen-deprived environments in which cancer cells reside. Now, using a multicellular, three-dimensional model system called a spheroid culture, Zhang et al. (Nature Communications, advance online publication February 18, 2014; DOI: 10.1038/ncomms4295) report the identification of a small molecule that is active against quiescent colon cancer cells. As opposed to monolayer cell cultures, the spheroids contain a periphery of proliferating cells that surround a core of quiescent cells. The authors screened 10,000 compounds and identified a molecule, referred to as VLX600, that was able to kill the cells in the core of the spheroids. Gene expression analysis in response to exposure to VLX600 suggested that the compound induces a glycolytic response that is dependent on expression of genes associated with low oxygen conditions. Further analysis indicated that exposure to VLX600 leads to an autophagic response, in which the cell degrades unnecessary components under conditions of nutrient stress. Moreover, VLX600 was found to inhibit mitochondrial oxidative phosphorylation, preventing production of ATP. Together, the data indicate that in contrast to typical chemotherapies that target fast growing cells, VLX600 exploits the metabolically compromised state of quiescent cancer cells. Finally, VLX600 exhibited antitumor activity in mouse colon cancer xenografts with minimal toxicity and acted synergistically with common chemotherapy drugs. This study offers compelling evidence for further investigation of VLX600 and related compounds in the treatment of cancer.
Reprinted with permission from O’Daniel, P. I., et al., J. Am. Chem. Soc., published online February 11, 2014, DOI: 10.1021/ ja500053x. Copyright 2014 American Chemical Society.
Methicillin-resistant Staphylococcus aureus (MRSA) is a global health threat, and there is a paucity of antibiotics that can effectively target this menacing pathogen. Its drug resistance stems from the acquisition of a gene encoding a penicillinbinding protein called PBP2a. PBP2a is not susceptible to the β-lactam antibiotics that are such effective inhibitors of other penicillin-binding proteins, and this resistance restores the ability of the bacteria to synthesize its cell wall. Potent inhibitors of PBP2a are needed to combat MRSA, and to this end, O’Daniel et al. (J. Am. Chem. Soc., published online February 11, 2014, DOI: 10.1021/ja500053x) report the discovery that certain oxadiazoles can effectively target PBP2a. In silico screening of 1.2 million compounds in complex with PBP2a of MRSA was performed to identify potential inhibitors of the enzyme. Twenty-nine compounds were selected for experimental testing in an antibacterial assay, and an oxadiazole with weak antibiotic activity was discovered. Additional screening of a small synthetic library of related oxadiazoles led to the identification of three compounds with promising antibiotic activity and minimal toxicity toward eukaryotic cells. Examination of the compounds in a mouse model of MRSA infection demonstrated that they had promising antibiotic activity and pharmacokinetic properties. Investigation into the mechanism of action of the compounds indicated that they do indeed inhibit cell wall synthesis. In addition, the compounds directly inhibited PBP2a in an in vitro binding assay. Collectively, the results highlight oxadiazoles as an exciting new class of PBP2a inhibitors that warrant further investigation for the treatment of drug-resistant bacterial infections.
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Eva J. Gordon, Ph.D.
FIGURING OUT FERROPTOSIS
Eva J. Gordon, Ph.D.
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GETTING TO THE MIDDLE OF CANCER
Reprinted from Cell, 156, Yang, W. S., et al., Regulation of Ferroptotic Cancer Cell Death by GPX4, 317−331. Copyright 2014, with permission from Elsevier.
Reprinted by permission from Macmillan Publishers Ltd.: Nature Communications, advance online publication, 18 February 2014, DOI: 10.1038/ncomms4295. © 2014 American Chemical Society
Published: March 21, 2014 585
dx.doi.org/10.1021/cb5001668 | ACS Chem. Biol. 2014, 9, 585−587
ACS Chemical Biology
Spotlight
Earlier experiments had suggested that a conserved palindromic region of the protein (AGAAAAGA, residues 113−120) could be important for prion disease. Antibodies that recognize this region of the protein can bind to normal HuPrPC but cannot access this region in the pathogenic HuPrPSc. At first, Abskharon et al. developed single domain antibodies, or nanobodies, to prion proteins. The nanobody that bound most tightly, Nb484, also reversibly inhibited the formation of amyloid fibrils in vitro and in mouse cells infected with scrapie. Nb484 bound to a region of the protein outside of the palindromic region but assisted with crystal packing. As a result, the team could get a crystal structure of the complex of HuPrP with Nb484. The resulting crystal structure showed that several residues in the conserved palindromic region formed an additional β-strand, which interacted with other β-strands to form a three-stranded antiparallel β-sheet. The new strand, β0, interacts with the existing β1 strand that has been implicated in conversion to PrPSc. In addition the hairpin that forms between these two strands makes several new hydrogen bonding sites available to solvent, providing a potential surface for intermolecular interactions. Although it is unclear why Nb484 halts prion formation, the nanobody may make the loop between one β-strand and α-helix more rigid. Stabilizing that structure could block the oligomer formation.
Ferroptosis is an iron-dependent mode of cell death that involves the production of lethal reactive oxygen species (ROS). This type of cell death is morphologically, biochemically, and genetically distinct from other types such as apoptosis, necrosis, and autophagy, but the molecular details of the process have not been elucidated. Now, Yang et al. (Cell, 2014, 156, 317−331) use metabolomic profiling and chemoproteomics to gain some insights into this largely unexplored mechanism for cell death. The authors exploit two small molecule inducers of ferroptosis, erastin and RSL3, in their approach. Erastin was used to probe the global changes in metabolism during ferroptosis. Cells treated with erastin exhibited reduced levels of glutathione and increased levels of lysophosphatidyl cholines, suggesting that erastin triggers formation of ROS and subsequent oxidative cell death. Further investigation revealed that erastin prevents cystine uptake, which leads to the depletion of glutathione and subsequent inactivation of glutathionedependent peroxidases. Interestingly, RSL3 also induces ferroptosis but in a glutathione-independent manner. To clarify its mechanism, fluorescent RSL3 derivatives were added to cells, and affinity purification and mass spectrometry were used to identify its molecular target. Remarkably, glutathione peroxidase 4 was identified as a target for RSL3, and the compound was shown to inhibit its peroxidase activity. In addition, examination of other small molecule inducers of ferroptosis showed that they also work through glutathione peroxidase 4 either like erastin, by depleting glutathione, or like RSL3, by inhibiting the enzyme directly. Finally, an analogue of erastin with improved pharmacological properties was shown to prevent tumor growth in mouse xenograft models, and B cell lymphomas and renal cell carcinomas were particularly sensitive to erastin and RSL3. Together, the data illuminate glutathione peroxidase 4 as a key regulator of ferroptosis and point to small molecule inducers of ferroptosis as potential anticancer agents.
Sarah A. Webb, Ph.D.
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I’LL HAVE THE LIVER
Eva J. Gordon, Ph.D.
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CLUES TO PRION OLIGOMER FORMATION
Reprinted by permission from Macmillan Publishers Ltd.: Nature, advance online publication, 23 February 2014, DOI: 10.1038/nature13020.
Over the past few years, researchers have developed proper care and feeding protocols to turn induced pluripotent stem (iPS) cells into a variety of cell types. Often, fibroblasts are first programmed to return to a stem state via the expression of a few known pluripotency genes. Now, a new method takes a shortcut from the norm, avoiding a return to full stem cell status. Zhu et al. (Nature, 2014; DOI: 10.1038/nature13020) adopted this approach because previous studies of iPS cells differentiated into liver hepatocytes resulted hepatocyte properties in vitro but failing to function like bona f ide adult hepatocytes in vivo. As an alternative to iPS cells, a population of more restricted, induced multipotent progenitor cells (iMPC) was produced. These cells could progress toward the endoderm lineage and ultimately into hepatocytes (iMPC-Heps). Each step in this process were enhanced by combinations of specific small moleucles. While characterizing the cells in a dish, the researchers found that their gene expression profile most closely matched that of human fetal hepatocytes. The cells could
Reprinted with permission from Abskharon, R. N. N. et al., J. Am. Chem. Soc. 136, 937−944. Copyright 2014 American Chemical Society.
Although researchers have long known that structural changes in the human prion protein (HuPrP) allow it to form pathogenic oligomers, the details have remained mysterious. A large segment of the N-terminal region of cellular HuPrP lacks a defined structure, making the protein difficult to crystallize and characterize. Using a single domain antibody as a coagent, Abskharon et al. have now crystallized full-length HuPrP (J. Am. Chem. Soc. 2014. 136, 937−944). Their structural studies indicate that changes in a short, conserved region of the protein may pave the way for oligomer formation. 586
dx.doi.org/10.1021/cb5001668 | ACS Chem. Biol. 2014, 9, 585−587
ACS Chemical Biology
Spotlight
proliferate from single colonies to large populations in vitro, an important prerequisite for most cell therapy approaches. The ultimate test came from transplanting the iMPC-Heps back into a mouse. The cells could repopulate a mouse model for liver disease and improve the survival of mice with chronic liver failure. Using laser-capture microscopy coupled with gene expression profiling, the transplanted cells were monitored and compared to adult hepatocytes. After proliferating inside of the mouse, the iMPC-Heps displayed very similar expression patterns to the adult hepatocytes, indicating that further maturation and specification occurs post-transplantation. This study shows that while the best place to grow liver cells is still in the liver, a new route to a potentially therapeutic cell type could help deliver the starting material of a healthy liver. Jason G. Underwood, Ph.D.
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dx.doi.org/10.1021/cb5001668 | ACS Chem. Biol. 2014, 9, 585−587
